Triptolide AttenuatesEndotoxin- andStaphylococcalExotoxin-InducedT-Cell Proliferation andProduction of Cytokinesand Chemokines
Teresa Krakauer,1 Xin Chen,2 O. M. Zack Howard,3
and Howard A. Young4
1Department of Immunology and Molecular Biology, United States Army MedicalResearch Institute of Infectious Diseases, Frederick, Maryland, USA2Basic Research Program, SAIC-Frederick, National Cancer Institute, Frederick,Maryland, USA3Laboratory of Molecular Immunoregulation, Frederick, Maryland, USA4Laboratory of Experimental Immunology, Center for Cancer Research, NationalCancer Institute-Frederick, Frederick, Maryland, USA
Proinflammatory cytokines mediate the toxic effects of superantigenic staphylococcalexotoxins (SE) and bacterial lipopolysaccharide (LPS). Triptolide, an oxygenatedditerpene derived from a traditional Chinese medicinal herb, Tripterygium wilfordii,inhibited SE-stimulated T-cell proliferation (by 98%) and expression of interleukin 1b,interleukin 6, tumor necrosis factor, gamma interferon, monocyte chemotactic protein1, macrophage inflammatory protein (MIP)-1a, and MIP-1b by human peripheral bloodmononuclear cells (PBMC). It also blocked the production of these cytokines andchemokines by LPS-stimulated PBMC in a dose-dependent manner. These resultssuggest that triptolide has potent immunosuppressive effects even counteracting the
Immunopharmacology and Immunotoxicology, 27:53–66, 2005
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ISSN: 0892-3973 print / 1532-2513 online
DOI: 10.1081/IPH-200051294
Address correspondence to Dr. Teresa Krakauer, Department of Immunology andMolecular Biology, United States Army Medical Research Institute of InfectiousDiseases, Bldg. 1425, Fort Detrick, Frederick, MD 21702-5011, USA; Fax: (301) 619-2348; E-mail: [email protected]
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4. TITLE AND SUBTITLE Triptolide attenuates endotoxin- and staphylococcal exotoxin-inducedT-cell proliferation and production of cytokines and chemokines,Immunopharmacology and Immunotoxicology 27:53 - 66
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6. AUTHOR(S) Krakauer, T Chen, X Howard, OMZ Young, HA
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14. ABSTRACT Proinflammatory cytokines mediate the toxic effects of superantigenic staphylococcal exotoxins (SE) andbacterial lipopolysaccharide (LPS). Triptolide, an oxygenated diterpene derived from a traditional Chinesemedicinal herb, Tripterygium wilfordii, inhibited SE-stimulated T-cell proliferation (by 98%) andexpression of interleukin 1beta, interleukin 6, tumor necrosis factor, gamma interferon, monocytechemotactic protein 1, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta by humanperipheral blood mononuclear cells (PBMC). It also blocked the production of these cytokines andchemokines by LPS-stimulated PBMC in a dose-dependent manner. These results suggest that triptolidehas potent immunosuppressive effects even counteracting the effects of superantigens and LPS. It also maybe therapeutically useful for mitigating the pathogenic effects of these microbial products bydownregulating the signaling pathways activated by both bacterial exotoxins and endotoxins.
15. SUBJECT TERMS Staphylococcal enterotoxin B, cytokines, lipopolysaccharide, chinese herb, triptolide
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effects of superantigens and LPS. It also may be therapeutically useful for mitigatingthe pathogenic effects of these microbial products by downregulating the signalingpathways activated by both bacterial exotoxins and endotoxins.
Keywords Cytokine, SEB, TSST-1, LPS, Immunosuppression, Triptolide.
INTRODUCTION
Staphylococcal exotoxins (SE) and bacterial lipopolysaccharide (LPS) are the
most common etiological agents causing shock.[1–3] Although these bacterial
products interact with host cells through different receptors, they both trigger
the release of inflammatory cytokines and chemokines, inducing inflammation
and resulting in tissue injury. LPS from Gram-negative bacteria binds directly
to CD14 that facilitates its interaction with Toll-like receptor 4 (TLR4), and
MD2 of monocytes/macrophages and other cells.[4] Subsequent transmem-
brane signaling then activates multiple pathways including the NF-kB and
p38 MAP kinase pathways resulting in cellular activation and expression of
inflammatory cytokines and chemokines. LPS induces excessive levels of the
proinflammatory cytokines, interleukin 1 (IL-1), and tumor necrosis factor a(TNFa); the key mediators of septic shock and more chronic inflammatory
reactions.[5]
Staphylococcal toxic shock syndrome toxin 1 (TSST-1) and the distantly
related staphylococcal enterotoxin A and B (SEA and SEB) also are potent
activators of the immune system and cause a variety of human diseases,
ranging from food poisoning to toxic shock.[1,2,6,7] These exotoxins bind to both
the major histocompatibility complex (MHC) class II molecules on antigen-
presenting cells and specific Vb regions of the T-cell antigen receptors.[8–10]
These toxins are called superantigens because of their ability to polyclonally
activate a considerable proportion of T cells.[8] Their interactions with cells of
the immune system also induce a massive production of proinflammatory
cytokines and chemokines.[10–12] The cytokines, TNFa, IL-1, and interferon
gamma (IFNg) are pivotal mediators in superantigen-induced toxic
shock.[2,7,10,13]
Both TNFa and IL-1 have potent immunostimulating activities and act
synergistically with IFNg to enhance inflammatory and immune reactions and
promote tissue injury.[14] Consequently, these cytokines are pathogenic at
high concentrations in vivo and are responsible for fever and toxic shock
induced by SE.[2,7,10]
Triptolide is a diterpenoid triepoxide isolated from the Chinese medicinal
herb Tripterygium wilfordii Hook F (TWHF). TWHF has been used for
centuries in traditional Chinese medicine to treat rheumatoid arthritis,
nephritis, and pulmonary diseases.[15] Extracts of TWHF suppress type II
collagen-induced arthritis and effectively prevent allograft rejection.[16,17] In
Krakauer et al.54
vitro, TWHF extracts inhibited T-cell activation by phytohemagglutinin
(PHA) or anti-CD3 antibody.[18] Triptolide has been identified as the major
active constituent responsible for the anti-inflammatory and immunosuppres-
sive effects of TWHF.[19 –21]
Triptolide has been reported to inhibit many biological processes in a wide
variety of cell types. Triptolide inhibits LPS-stimulated COX-2 mRNA and
synthesis of PGE2 in LPS-stimulated monocytes.[22]
In human synovial fibroblasts, triptolide suppresses the production and
expression of prometalloproteinases 1 and 3 and inhibits the expression of
COX-2 and IL-1-induced PGE2 production.[23] Triptolide also inhibits vascular
endothelial cell growth factor expression in phorbol 12-myristate 13-acetate
(PMA)-activated endothelial cells[24] and attenuates the expression of IL-6, IL-
8, and cell adhesion molecule ICAM-1 by PMA-stimulated human bronchial
epithelial cells.[25] The effects of triptolide on other cell types include the
inhibition of the expression of C3, CD40, and B7H in TNFa-activated human
proximal tubular epithelial cells[26] and suppression of LPS-induced TNFa, IL-
1b, and nitric oxide production by microglial cells.[27] Additionally, triptolide
inhibits T-cell IL-2 expression at the purine-box/NF-AT and NFkB target
sequence after specific DNA binding.[28]
A proposed mechanism of action for triptolide is inhibition of NFkB
transcriptional activation. Additional studies indicated that triptolide also
blocks constitutive expression of cell-cycle regulators, cyclins D1, B1, and A1
in bronchial epithelial cells.[25] Triptolide also has antineoplastic activity and
sensitizes cells to TNFa-induced apoptosis in tumor cells via the activation of
caspase 3.[29,30] Recently, cDNA array analysis indicated that triptolide
inhibits the expression of genes associated with cellular inflammation, cell-
cycle progression, and cell survival.[31] A soluble derivative of triptolide
(PG490-88) was effective in suppressing obliterative airway disease in a
mouse allograft model and blocks bleomycin-induced lung fibrosis.[32]
This study was undertaken to determine the effect of triptolide on
staphylococcal superantigen-induced T-cell activation and cytokine production
by human peripheral blood mononuclear cells (PBMC) to determine whether it
may be used to suppress toxic shock syndrome. These effects were compared
with those of triptolide on LPS-stimulated PBMC, as previous studies used
LPS or cytokines as the stimulating agents.
MATERIALS AND METHODS
ReagentsPurified TSST-1 and SEB were obtained from Toxin Technology (Sarasota,
FL, USA). The endotoxin content of these preparations was < 1 ng of
endotoxin/mg protein as determined by the Limulus amoebocyte lysate
55Triptolide May Treat Toxic Shock
gelation test (BioWhittaker, Walkersville, MD, USA). Human (h) recombinant
(r) TNFa, antibodies against hTNFa, peroxidase-conjugated antirabbit IgG,
and peroxidase-conjugated antigoat IgG were obtained from Boehringer-
Mannheim (Indianapolis, IN, USA). Human rIFNg and rIL-6 were obtained
from Collaborative Research (Boston, MA, USA). Antibodies against IFNg and
MCP-1 were obtained from BDPharMingen (San Diego, CA, USA). Recombi-
nant MCP-1, MIP-1a, MIP-1b, and antibodies against IL-1b, IL-6, MIP-1a, and
MIP-1b were purchased from R&D Systems (Minneapolis, MN, USA).
Lipopolysaccharide (Escherichia coli 055:B5) was purchased from Difco
(Detroit, MI, USA). Triptolide was obtained from Calbiochem (San Diego,
CA, USA) and dissolved in DMSO. All other common reagents were from
Sigma (St. Louis, MO, USA).
Cell CultureHuman PBMC were isolated by Ficoll-Hypaque density gradient centri-
fugation of heparinized blood from normal human donors. PBMC were
cultured at 37�C in RPMI 1640 medium supplemented with 10% fetal bovine
serum at a concentration of 106 cells/mL in 24-well plates. Cells were
stimulated with TSST-1 (200 ng/mL), SEB (200 ng/mL), or LPS (5 ng/mL) for
16 hr. Various concentrations of triptolide were added simultaneously with
TSST-1, SEB, or LPS. Supernatants were harvested and analyzed for IL-1b,
TNFa, IL-6, IFNg, MCP-1, MIP-1a, and MIP-1b. Cytotoxicity was measured by
the uptake of trypan blue.
T-cell proliferation was assayed with PBMC (105 cells/well) that were
plated in triplicate with TSST-1 or SEB (200 ng/mL), with or without
triptolide, for 48 hr at 37�C in 96-well microtiter plates. Cells were pulsed with
1 mCi/well of [3H]thymidine (New England Nuclear, Boston, MA, USA) during
the last 5 hr of culture as described previously.[33] Cells were harvested onto
glass fiber filters, and incorporation of [3H]thymidine was measured by
liquid scintillation.
Cytokine AssaysCytokines and chemokines were measured by an enzyme-linked immu-
nosorbent assay (ELISA) with cytokine- or chemokine-specific antibodies as
previously described.[33,34] Human recombinant cytokines and chemokines
(20–1000 pg/mL) were used as standards for calibration on each plate. The
detection limit of each assay was 20 pg/mL.
Ribonuclease AssaysTotal RNA was isolated 4 hr after SE or LPS treatment from cells by
using a guanidinium isothiocyanate/chloroform-based technique (TRIZOL,
Krakauer et al.56
GIBCO, Grand Island, NY, USA) per the manufacturer’s instructions. The
RNase protection assay was performed as follows: total cellular RNA (5–
10 mg) was hybridized with a 33-P UTP-labeled RNA probe (mck-1, mck-2b,
mck-3b, mck-5 utilizing the BDPharmingen RiboQuant In Vitro Transcrip-
tion kit, 1 � 106 cpm/RNA sample) using the BDPharmingen hybridization
buffer, according to the manufacturer’s directions (BDPharmingen). After
hybridization, the samples were treated with RNase A and T1 according
to the procedure provided by BDPharmingen; the RNase was inactivated;
and the protected RNA was precipitated with a master cocktail containing
200 mL of Ambion (Austin, TX, USA) Rnase inactivation reagent, 50 mL of
ethanol, 5 mg of yeast tRNA, and 1 mL of Ambion GycoBlue co-precipitate
per RNA sample. The samples were mixed well, incubated at � 70�C for
30 min, and centrifuged at 14,000 rpm for 15 min at room temperature.
The pellets were resuspended in 3 mL of BDPharmingen sample buffer and
subjected to polyacrylamide gel electrophoresis as recommended by the
manufacturer (BDPharmingen).
Statistical AnalysisData were expressed as the mean ± SD and were analyzed by the
Student’s t-test with Stata (Stata Corp., College Station, TX). Differences
between triptolide-treated groups and untreated controls were considered
significant if p was < .05.
RESULTS
Triptolide Blocked Cytokine and Chemokine ProductionBased on reports that triptolide has anti-inflammatory effects, we tested
its potency in blocking cytokine and chemokine production by two different
stimulants, the superantigen TSST-1 and LPS. Figure 1A shows that
triptolide blocked the production of IL-1b and IL-6 in TSST-1-stimulated
PBMC in a dose-dependent manner. A low dose of triptolide (10 nM) reduced
the IL-1b and IL-6 levels to 12% and 17% in culture supernatants,
respectively. The production of other inflammatory cytokines (TNFa and
IFNg) and chemokines (MCP-1, MIP-1a, MIP-1b) also were blocked by
triptolide (Fig. 1B and 1C). Higher concentrations of TSST-1 (500 ng/mL)
failed to reverse the suppressive effects of triptolide. Dose response inhibition
curves of triptolide were similar at both high TSST-1 (1000 ng/mL) and low
TSST-1 (10 ng/mL) concentrations (data not shown).
The suppressive effects of triptolide were further examined by using
LPS as a stimulant that activates a different receptor. Triptolide also inhibited
57Triptolide May Treat Toxic Shock
Figure 1: Dose-response inhibition of (A) IL-1b and IL-6, (B) TNFa and IFNg, (C) MCP-1, MIP-1a,and MIP-1b production by PBMC stimulated with 200 ng/mL of TSST-1 in the presence ofvarious concentrations of triptolide. Values represent the mean ± SD of duplicate samplesand results represent three experiments. Results are statistically significant (p < .05) betweenTSST-1 and TSST-1 plus triptolide samples at concentrations of 1 to 100 nM of triptolide for IL-1b,IL-6, and MCP-1. For TNFa, IFNg, MIP-1a, and MIP-1b results are statistically significant (p < .05)between TSST-1 and TSST-1 plus triptolide samples at 10 to 100 nM.
Krakauer et al.58
IL-1b, IL-6, and TNFa production by LPS-stimulated PBMC dose-dependent-
ly, reducing IL-1b, IL-6, and TNFa by 49%, 58%, and 50%, respectively, at 10
nM of triptolide (Fig. 2A and 2B). Higher concentrations of triptolide blocked
the production of these cytokines and the chemokines, MIP-1a and MIP-1b,
by LPS-activated cells more completely, whereas MCP-1 production was
totally inhibited at 10 nM of triptolide. Triptolide did not affect the viability of
Figure 2: Inhibition of (A) IL-1b, IL-6, and TNFa; (B) MCP-1, MIP-1a, and MIP-1b production byPBMC stimulated with 5 ng/mL of LPS in the presence of various concentrations of triptolide.Values represent the mean ± SD of duplicate samples and results represent three experi-ments. Results are statistically significant (p < .05) between LPS and LPS plus triptolide samplesat concentrations of 1 to 100 nM of triptolide for all except MIP-1a and MIP-1b. Results arestatistically significant (p < .05) between LPS and LPS plus triptolide samples at 10 to 100 nMfor MIP-1a and MIP-1b.
59Triptolide May Treat Toxic Shock
the cells over the concentration range used in these studies (1–30 nM), as
confirmed by trypan blue dye exclusion test. However, at 100 nM triptolide,
20% of PBMC took up trypan blue stain after 48 hr.
Figure 3 compares the inhibition by 10 nM triptolide of cytokine and
chemokine production by PBMC cultures stimulated with another staphylo-
coccal exotoxin, SEB, with the effects of TSST-1 and LPS. The inhibition of
SEB-stimulated cells was similar to that of TSST-1 suggesting that triptolide
is an effective inhibitor of the superantigen-activated pathways.
Figure 3: Inhibition of (A) IL-1b, IL-6, TNFa; and IFNg; and (B) MCP-1, MIP-1a, and MIP-1bproduction by PBMC stimulated with TSST-1 (200 ng/mL), SEB (200 ng/mL), or LPS (5 ng/mL) inthe presence of 10 nM of triptolide. Values represent the mean ± SD of PBMC cultures from 6blood donors. Results are statistically significant (p < .05) between stimulant (TSST-1, SEB, orLPS) and stimulant plus triptolide samples.
Krakauer et al.60
Triptolide Inhibited TSST-1- and LPS-Induced Cytokineand Chemokine mRNA ExpressionWe sought to determine the mechanism of inhibition of superantigen
and LPS-induced cytokines and chemokines by triptolide at the molecular
level. Total RNA was extracted from stimulated cells 4 hr after triptolide
Figure 4 (A) and (B): Cytokine and chemokine mRNA analysis. Total RNA was extractedfrom human PBMC treated for 4 hr with 200 ng/mL of TSST-1 or 5 ng/mL of LPS in the presenceor absence of triptolide. Multiprobe RNase protection analysis was performed as described inMaterials and Methods using 5 mg of total RNA per lane. Lanes 1, 2, 3, and 4 represent cells inmedium alone, TSST-1-stimulated cells, TSST-1-stimulated cells plus 5 nM of triptolide, and TSST-1-stimulated cells plus 10 nM of triptolide, respectively. Lanes 5, 6, and 7 represent LPS-stimulated cells, LPS-stimulated cells plus 10 nM of triptolide, and LPS-stimulated cells plus30 nM of triptolide. Data shown are representative of experiments repeated three or moretimes. The cytokine or chemokine tested is shown to the left of each RPA; Ltn (lymphotoxin),Rantes (CCL5), IP10 (CXCL10), MIP-1b (CCL4), MIP-1a (CCL3), MCP-1 (CCL2), IL-8 (CXCL8),TNFa, IL-1a and IFNg.
61Triptolide May Treat Toxic Shock
treatment and gene expression was measured with a Multiprobe RNase
protection assay. L32 rRNA and GAPDH RNA were used as internal
standards for RNA measurements. Figure 4A and 4B show that 5 and 10 nM
of triptolide blocked TSST-1-mediated increases in the RNA for TNFa, IL-1b,
IL-8, IFNg, IP-10, MIP-1a, MIP-1b, and MCP-1. At this dose of triptolide,
LPS-induced expression of most of the RNA examined was partially blocked.
A higher concentration of triptolide (30 nM) further reduced the LPS-
mediated mRNA expression of TNFa, IL-1b, IL-8, IFNg, IP-10, MIP-1a, MIP-
1b, and MCP-1.
Triptolide Inhibited Superantigen-InducedT-Cell ProliferationBecause superantigen polyclonally activates T cells, the effect of triptolide
on SE-induced T-cell proliferation was next investigated. Figure 5 shows that
triptolide is a potent inhibitor: reducing SEB- and TSST-1-stimulated T-cell
proliferation in a dose-dependent manner and achieving 98% inhibition at
10 nM of triptolide.
DISCUSSION
Shock caused by bacterial products from both Gram-positive and Gram-
negative bacteria is a serious clinical problem and inhibition of any single
cytokine by a specific cytokine antibody or receptor antagonist often does not
Figure 5: Inhibition of T-cell proliferation in PBMC stimulated with 200 ng/mL of TSST-1 or SEB inthe presence of various concentrations of triptolide. Values represent the mean ± SD oftriplicate samples and results represent three experiments. Results are statistically significant(p < .05) between stimulant (TSST-1 or SEB) and stimulant plus triptolide samples.
Krakauer et al.62
result in successful treatment and recovery. Anti-inflammatory and im-
munosuppressive therapeutics represent a potentially useful treatment
independent of the inciting agents by targeting common downstream
signaling pathways affecting multiple cytokines and chemokines. The results
presented here indicate that triptolide suppressed the induction of proin-
flammatory cytokines and chemokines by TSST-1-, SEB-, and LPS-sti-
mulated human mononuclear cells. The production of these mediators by
monocytes/macrophages and T cells in response to superantigens and LPS
initiates leukocyte activation and migration, contributing directly to in-
flammation and tissue injury associated with shock. T-cell proliferation also
was blocked by triptolide.
Previous studies showed that triptolide inhibited transcriptional activa-
tion through NFkB[28] and suppressed TNFa production by LPS-stimulated
macrophages[23] and IL-2 production by PHA-activated T cells.[28] Our results
extend these observations by showing inhibition of multiple inflammatory
mediators in staphylococcal exotoxin-activated PBMC at both transcriptional
and protein level. Genes for proinflammatory mediators IL-1 and TNFacontain DNA binding sequences for the transcriptional factor NF-kB, and
triptolide was shown to inhibit the activation of IL-2 transcriptional factors.[28]
Attenuated T-cell activation with decreased elaboration of key proin-
flammatory cytokines by triptolide suggests triptolide may prove useful in
treating superantigen-induced shock. Multiple clinical trials of extracts of
TWHF in rheumatoid patients indicate that triptolide is the active component
responsible for the immunosuppressive effects (reviewed in Ref. [15]). Oral
and intraperitoneal administration of triptolide in both mice and rats at doses
of up to 0.25 mg/Kg for prolonged periods of 3 to 4 weeks produce no lethal
effects,[16,17,35,36] although infertility is a known side effect.[37]
Our studies showed that triptolide suppresses a broad range of cytokine
production induced by superantigens and LPS, suggesting that triptolide
targets several intracellular signaling pathways. One prominent pathway is
the transcriptional activation of NF-kB that regulates the expression of
inflammatory cytokines, cyclooxygenase 2, and cell adhesion molecules. This
interference of NF-kB activation by triptolide likely accounts for its potent
immunosuppressive effects. In conclusion, due to the broad spectrum of
cytokines antagonized, and based on its beneficial therapeutic effects in
autoimmune diseases, triptolide may prove useful as a therapeutic for the
treatment of toxic shock.
ACKNOWLEDGMENTS
We thank Marilyn Buckley for excellent technical assistance and Lorraine
Farinick for preparation of illustrations.
63Triptolide May Treat Toxic Shock
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